Summary of Work: To develop methods for in vivo imaging of CNS receptor and signal transduction mechanisms, radiolabeled ligands with appropriate SAR requirements are being produced and used to develop in vivo animal models, and will lead ultimately to studies in human subjects. Synthetic organic chemistry techniques are used to produce cold labeled derivatives of candidate compounds to determine SAR in displacement assays using standard beta (C-14 or H-3) or gamma (I-125) emitting radioligands. If cold labeled compounds retain activity, techniques are developed for rapid radiosynthesis. Positron (F-18, C-11, N-13) or single photon (I-123, In-111, Tc-99m) emitting derivatives are used in initial in vivo studies to determine blood brain barrier penetration and in vivo activity; both autoradiographic and external imaging (high resolution small animal PET) techniques are used. Tracer kinetic (bolus or equilibrium) models have been developed in rodents and primates, and validated using activation and displacement techniques. A cold fluorinated (fluoroethyl-carbamate) derivative of forskolin was previously synthesized, and high affinity (Kd < 40 nM), saturable binding to adenylyl cyclase demonstrated in vitro. A tritiated derivative of this compound has now been shown to cross the blood brain barrier in sufficient concentration (> 1% of injected dose/g) when administered in vivo to rodents to allow PET imaging when labeled with F-18. The tritiated compound accumulated in brain areas following in vivo infusion at a rate proportional to regional adenylyl cyclase concentration. The binding of the labeled forskolin to isoforms type II and V has additionally been shown to be sensitive to activation of the enzyme by Gs-alpha-GTP. These features will be capitalized upon to develop tracer kinetic models, ultimately permitting dynamic imaging of adenylyl cyclase activity in vivo using PET. Further studies to determine how the binding of fluoroethyl-carbamate forskolin to the various cyclase isoforms might be similarly affected by calcium-calmodulin, PKC, and beta-gamma subunits of the G-protein complex are underway. A multi-animal study of the time activity distribution of the triatiated compound is presently being analyzed. Results of the above studies are being prepared for publication. Pilot studies evaluating voltage sensitive compounds for use in PET have been completed.